- LH is an essential enzyme in collagen biosynthesis which hydroxylates the lysine residues required for the formation of intermolecular crosslinks that are critical for the tensile strength of the collagen fibrils. Patients with the inherited connective tissue disorder EDS type VI (EDS VI) have clinical features of hyperextensible skin and joints, fragile skin, and kyphoscoliosis. Patients with EDS VIA are biochemically characterized as deficient in LH; this enzyme deficiency results from different mutations in the originally identified LH gene (LH1). In contrast, the subclass of patients with EDS VIB have normal levels of LH activity and the biochemical basis for this disorder is unknown. Recently, two isoforms of LH (LH2 and LH3), together with an alternatively spliced form LH2alt, have been reported that are 75% similar to LH1. The principal investigator hypothesizes that these isoforms have different functions from LH1; whereas LH1 hydroxylates lysine residues in helical collagen chains, LH2 and/or LH3 synthesize hydroxylysyl residues in the nonhelical telopeptide region. Since the nonhelical hydroxylysyl residues determine the stability of collagen crosslinks, LH2 or LH3 may be prime targets for mutations producing the clinical phenotype of EDS VIB. She will use a baculovirus vector to express LH2, LH2alt, and LH3 in insect cells, and their potential to hydroxylate lysines in helical and nonhelical synthetic peptides will be examined. This hydroxylation pattern will then be examined in an optimized baculovirus system in which recombinant type III collagen is expressed together with each LH isoform. A fetal cell line in which the telopeptide lysines are highly hydroxylated will be screened for a potentially new telopeptide LH. The biochemical basis for EDS VIB, in which cells from these patients have hydroxylysine-deficient collagen but apparently normal LH activity, will be analyzed for changes in lysyl hydroxylation and collagen crosslinking which may suggest deficiency of one of the LH isoforms. Sequencing of selected cDNAs may identify mutations in the LH genes responsible for EDS VIB. This study should provide insight into disorders associated with aberrant crosslinking in the extracellular matrix and may suggest the design of therapeutic agents to modify abnormal levels of the LH isoform(s) that may affect the strength of the connective tissue.